J. FOR. SCI., 55, 2009 (10): 461–468 461 JOURNAL OF FOREST SCIENCE, 55, 2009 (10): 461–468 Acacia catechu Willd. is an medicinally important leguminous tree that grows naturally in all kinds of geological formations and soils of all kinds. Katha, which is an important ingredient in the “Pan” preparation commonly used in northern India, is commercially obtained from this plant. A. catechu has better microflora in its rhizosphere. e arbus- cular mycorrhizal (AM) fungi take an important ecological position amongst various microorganisms colonizing the rhizosphere of plants. e occurrence of endomycorrhizal fungi (AM) in soil, their asso- ciation with both forestation and agricultural crops are well documented (R et al. 1999; G, S 2002). Inoculation with a suitable AM fungal strain to improve the growth and survival of plant seed- lings in forestation is very essential. e role of AM fungi in improving the quality and survival of plant seedlings after plantation has been well recognized (K et al. 1992; K, U 1999; O 2003; B et al. 2007). e mutual relation- ship between mycorrhizal fungi and host plants has been studied in terms of the benefits to individual plants and fungi (F, R 1995; S, S 1996). e effect of mycorrhizal fungi on nutrient uptake and plant growth has been exten- sively studied (T, B 1997; W-  et al. 1999; L et al. 2000; A-H, A-M 2006). Mycorrhizal infection has a particular value for legumes because nodulation and symbiotic nitrogen fixation by Rhizobia require an adequate phosphorus supply and a restricted root system leads to poor competition for soil phospho- rus (C et al. 1978). Various researchers have made efforts to increase the quality of seedlings of forest trees through the inoculation of a suitable mycorrhizal strain alone Diversity of endomycorrhizal fungi and their synergistic effect on the growth of Acacia catechu Willd. V. P 1 , A. A 2 1 Rain Forest Research Institute (ICFRE), Assam, India 2 Botany Department, Kurukshetra University, Haryana, India ABSTRACT: e diversity of arbuscular mycorrhizal (AM) fungi of Acacia catechu Willd. was studied. Dominant AM spores, the bacterium Rhizobium sp. along with the fungus Trichoderma viride were isolated from the rhizosphere of A. catechu and mass-produced in laboratory. e co-inoculation effect of Glomus mosseae, Glomus fasciculatum, mixed AM (Glomus spp. [except G. mosseae, G. fasciculatum] with Acaulospora spp., Sclerocystis spp. and Gigaspora spp.), Rhizobium sp. and Trichoderma viride was studied as exerted on the growth of A. catechu seedlings. All inoculated seedlings showed improved seedling growth compared to the control. Inoculated seedlings had a pronounced effect on all growth parameters such as height, fresh and dry weight of roots and shoots, AM spore count, per cent mycor- rhizal colonization in roots and root nodule number in comparison with uninoculated seedlings. Phosphorus uptake was also higher in inoculated seedlings than in the control. is study provides a good scope for commercially utiliz- ing the efficient strains of AM fungi for beneficial effects with other beneficial rhizosphere microflora in the primary establishment of slow growing seedlings ensuring better survival and improved growth. Keywords: Acacia catechu Willd.; co-inoculation; phosphorus; Rhizobium sp.; Trichoderma viride; AM diversity Abbreviations: ppm – parts per million, LSD – least standard deviation, sp. – species, AM – arbuscular mycorrhiza 462 J. FOR. SCI., 55, 2009 (10): 461–468 Table 1. Growth response of Acacia catechu to double inoculation after 90 days S. No. Treatment (double inoculation) Increase in height (cm) Root length (cm) Fresh shoot wt. (g) Dry shoot wt. (g) Fresh root wt. (g) Dry root wt. (g) VAM spore number/50 g soil Mycorrhizal colonization in roots (%) Presence of nodules 1 Control *1.5 ± 0.03 13.0 ± 0.70 6.8 ± 0.01 5.73 ± 0.02 8.4 ± 0.34 6.62 ± 0.36 39 ± 0.70 56.66 ± 2.72 _ 2 Mixed AM + Rhizobium sp. 22.0 ± 1.06 21.0 ± 0.70 7.9 ± 0.03 6.37 ± 0.02 10.6 ± 0.69 7.65 ± 0.26 113 ± 2.82 93.33 ± 5.44 ++ 3 G. mosseae + Rhizobium sp. 22.2 ± 1.41 22.5 ± 1.76 10.3 ± 0.36 7.89 ± 0.23 14.3 ± 0.40 10.09 ± 5.65 120 ± 2.12 93.33 ± 4.61 +++ 4 G. fasciculatum + Rhizobium sp. 8.25 ± 0.35 20.0 ± 1.41 7.7 ± 0.47 6.20 ± 0.35 8.8 ± 1.08 6.56 ± 0.47 102 ± 0.70 80.55 ± 2.26 + 5 Trichoderma viride + Rhizobium sp. 4.0 ± 0.35 17.5 ± 5.30 7.2 ± 0.42 6.01 ± 0.15 8.5 ± 0.61 6.81 ± 0.13 101 ± 1.41 76.66 ± 2.72 – *Mean of five replications each, ± standard error, +++ abundant, ++ moderate, + low, – absent Source of variation Degree of freedom Sum of squares Mean of squares F obtained (F ratio) F crit. (table) Between 4 441 110.25 15.7* 0.05 P = 5.19 Within 5 32 6.4 0.01 P = 11.3 Total 9 473 *According to the analysis of variance (ANOVA) of data, the calculated F ratio is 15.7, which is significant at 0.01% P level (F crit. 0.01 = 5.19, 0.05 = 11.39) J. FOR. SCI., 55, 2009 (10): 461–468 463 or AM-Rhizobium sp. combinations in nursery conditions (A, R 1990; B et al. 1990; C 1990; R, B 1990; L, H 1991; T et al. 1992; S, T 1997; R et al. 1999; K-  et al. 2002; P et al. 2005; K, Z 2007). But very little or hardly any work has been done to improve and produce the quality seedlings of A. catechu by using the triple co-inoculation of VAM strains since this plant is economically more important on a local level and slow growing as well. Bearing this in mind, the present study was under- taken to determine the effect of double and triple inoculation (co-inoculation) of AM fungi with other rhizospheric microflora on the growth performance of A. catechu. MATERIALS AND METHODS e native predominant AM fungi, i.e. Glomus mosseae and Glomus fasciculatum, were isolated from the rhizosphere of A. catechu. All the remaining species of Glomus (except G. mosseae, G. fascicula- tum), Acaulospora, Sclerocystis and Gigaspora were also isolated and mixed together to prepare a mixed AM inoculum. Rhizobium sp. and Trichoderma viride were also isolated from the rhizosphere of A. catechu in a similar way. All mycorrhizal inocula (Glomus mosseae, Glomus fasciculatum, Mixed AM) were mass-produced on maize (Zea mays L.). Rhizobium sp. inoculum was mass-cultured on yeast extract mannitol agar (YEMA) medium. T. viride was also mass-produced on wheat bran:saw dust:water (3:1:4) medium for further inoculation experiments. Seedlings of A. catechu were procured from Divi- sional Forest Nursery, Hamirpur Forest Department, Himachal Pradesh, India. Inoculation: Inoculation experiments were de- signed in double and triple combinations (co-inocu- lation). In double inoculation, mixed AM (except G. mosseae and G. fasciculatum), G. mosseae, G. fas- ciculatum and Trichoderma viride were mixed with Rhizobium species only while in triple inoculation, mixed AM (except G. mosseae and G. fasciculatum), G. mosseae and G. fasciculatum were mixed together with T. viride and Rhizobium species. All inocula- tion experiments were performed in sterilized soils. Two seedlings of A. catechu were grown in each ex- perimental earthen pot (30 × 30 cm) in a sandy soil mixture (300:1,500 g). To each pot 10% of the fungal (T. viride), bacterial (Rhizobium sp.) and mycor- rhizal inocula along with infected roots were added around the rhizosphere. Only five replications were prepared. In control sets, no inoculum was added. After 45 and 90 days interval observations were re- corded on seedling shoot length (increase in height), root length, shoot and root fresh and dry weight, percentage mycorrhizal colonization in roots, AM spore number, root nodule number and phosphorus content (%) of seedlings. But only the observations after 90 days are cited in this paper. Percentage mycorrhizal colonization in roots was studied (P, H 1970). e AM spore quanti- fication was determined (G, N 1963). Phosphorus content (%) of shoots and roots was determined by vanadomolybdate phosphoric yellow colour method (J 1973). Data were compared with the control after treatment. e data were analyzed statistically by the analysis of variance (ANOVA) (P 2000). RESULTS AND DISCUSSION Four genera of VAM fungi, e.g. Glomus, Acau- lospora, Gigaspora and Sclerocystis, have been reported from the rhizosphere of A. catechu. Entrophospora and Scutellospora were found absent in the rhizosphere. Glomus spp. were present in abundance compared to the other genera. e per- centage mycorrhizal colonization in roots was (90 ± 7.07) and VAM spore number was (182.5 ± 5.30) in 50 g of soil (five random replications). Both arbus- cular and vesicular types of colonization were found in roots of A. catechu. e AM fungi present in the rhizosphere of A. catechu were Glomus mosseae, G. fasciculatum, G. intraradices, G. macrocarpum, Acaulospora laevis, A. foveata, Gigaspora sp., Scle- rocystis coremioides. Double inoculation: e effect of double inocu- lation after 90 days on Acacia catechu is shown in Table 1. It was revealed that the G. mosseae + Rhi- zobium sp. combination showed a maximum sig- nificant (F ratio 15.7, P > 0.05) increase in height (22.2 ± 1.41) compared to the other treatments. AM spore numbers, percentage mycorrhizal coloniza- tion in roots, increase in plant height, fresh and dry weights (shoot and root) were increased with the time period when compared with earlier data of 45 days. e presence of nodules was also high in G. mosseae and Rhizobium sp. treatment (Table 1). No nodule was present either in the control or in Trichoderma viride + Rhizobium sp. treatments. Triple inoculation: e response of A. catechu to triple inoculation after 90 days is shown in Table 2. It is evident from the table that the G. mosseae + Rhizobium sp. + Trichoderma viride (triple inocu- lum) combination showed a maximum significant (F ratio 48.0, P > 0.05) increase in height, root length, 464 J. FOR. SCI., 55, 2009 (10): 461–468 Table 2. Growth response of Acacia catechu to triple inoculation after 90 days S. No. Treatment (triple inoculation) Increase in height (cm) Root length (cm) Fresh shoot wt. (g) Dry shoot wt. (g) Fresh root wt. (g) Dry root wt. (g) VAM spore number/50 g soil Mycorrhizal colonization in roots (%) Presence of nodules 1 Control *1.5 ± 0.03 13.0 ± 0.70 6.8 ± 0.01 5.73 ± 0.02 8.4 ± 0.34 6.62 ± 0.36 39 ± 0.70 56.66 ± 2.72 _ 2 Mixed AM + Rhizobium sp.+ Trichoderma viride 11.2 ± 0.70 24.0 ± 5.65 9.9 ± 1.14 7.61 ± 0.74 13.7 ± 2.03 9.65 ± 0.95 118 ± 3.53 80.0 ± 4.71 ++ 3 G. mosseae + Rhizobium sp. + Trichoderma viride 22.2 ± 0.03 31.0 ± 6.36 10.6 ± 0.95 7.85 ± 0.53 20.5 ± 3.39 13.08 ± 2.09 126.5 ± 4.59 100.0 ± 0 +++ 4 G. fasciculatum + Rhizobium sp. + Trichoderma viride 5.88 ± 1.41 19.0 ± 0.70 7.8 ± 0.35 6.37 ± 0.20 10.4 ± 5.65 7.82 ± 0.01 95 ± 3.53 83.33 ± 2.72 + *Mean of five replications each, ± standard error, ++ moderate, +++ abundant, + low, – absent Source of variation Degree of freedom Sum of squares Mean of squares F obtained (F ratio) F crit. (table) Between 3 288 96 48.0* 0.05 P = 3.11 Within 4 8 2 0.01 P = 5.06 Total 7 295 *According to the analysis of variance (ANOVA) of data, the calculated F ratio is 48.0, which is significant at 0.01% P level (F crit. 0.01 = 6.59, 0.05 = 16.69) J. FOR. SCI., 55, 2009 (10): 461–468 465 fresh and dry weights of shoot and root, AM spore number and percentage mycorrhizal colonization in roots after 90 days in comparison with the other inoculated treatments and control in which all these above-mentioned growth parameters were low. In this case, the G. mosseae + Rhizobium sp. + Tri- choderma viride treatment also had more nodules than the other treatments. Nodules were absent in the control. Double inoculation of mixed AM + Rhizobium sp. also showed a higher P content (%) in shoots (0.48 ± 0.01) and roots (0.99 ± 0.01) than the other treatments as well as the control (Table 3) whereas the triple inoculation of mixed VAM + Rhizobium sp. + Trichoderma viride showed a higher P content of roots (0.99 ± 0.01) than all the other treatments and control. P content of shoots (0.48 ± 0.01) was higher in all treatments than in the control and it was also equal to the G. mosseae + Rhizobium sp. + Trichoderma viride treatment (0.48 ± 0.01). It is evident from the results that all the inoculations had a higher P content of shoots and roots than the con - trol. P content of roots was higher than P content of shoots in all the inoculated seedlings of A. catechu. ere are many reports that suggest the impor- tance of VAM fungi in producing quality seedlings. Recently, papers were published in which the positive growth effect and nutrient uptake by plants through AM inoculation was seen under different water regimes and bioremediated and agricultural soils (A-H, A-M 2006; A Z et al. 2009). J et al. (2007) studied the effect of AM inoculation on the drought resist- ance of wild jujube (Ziziphus spinosus Hu) seedlings. ey found that arbuscular mycorrhizal fungi helped withstand the water stress and also improved the growth of the seedlings. In the present study, a soil-based inoculum was used for all the experiments. Hence, the better growth responses were seen in A. catechu. This might be due to higher reproduction of AM fungi present in the soil-based inoculum, which sprouted rapidly from extracellular and intracellular hyphae present in the soil and root inoculum. Co-inocula- tion has a synergistic effect on seedlings through increasing the efficiency of the shoot and root system in providing the plant with essential levels of P and N for growth. Inoculated seedlings with the entire test AM fungi increased the phosphorus content of roots and shoots as compared to the control in this study. e increased rate of phosphorus uptake and inflow in roots has been regarded as the major contribution of AM infection (M 1973). Present findings also indicated that mycorrhizal co-inoculated seedlings Table 3. Effect of co-inoculation on the phosphorus content of Acacia catechu S. No. Treatment Phosphorus content (%) (ppm) *shoot *root Double inoculation 1 Mixed AM + Rhizobium sp. 0.48 a ± 0.01 0.99 d ± 0.01 2 G. mosseae + Rhizobium sp. 0.46 a ± 0.02 0.91 d ± 0 3 G. fasciculatum + Rhizobium sp. 0.44 a ± 0 0.79 c ± 0.01 4 Trichoderma viride + Rhizobium sp. 0.43 a ± 0.02 0.61 b ± 0.01 5 Control 0.41 a ± 0 0.43 a ± 0 LSD 0.37 0.12 Triple inoculation 1 Mixed AM + Rhizobium sp. + Trichoderma viride 0.48 a ± 0.01 0.99 b ± 0.01 2 G. mosseae + Rhizobium sp. + Trichoderma viride 0.48 a ± 0 0.89 b ± 0.021 3 G. fasciculatum + Rhizobium sp. + Trichoderma viride 0.46 a ± 0.023 0.88 b ± 0.010 4 Control 0.41 a ± 0 0.43 a ± 0 LSD 0.25 0.13 *Average of five replications each, means followed by the same superscript letter in columns are not significant at 0.05 P level, ± standard error of mean 466 J. FOR. SCI., 55, 2009 (10): 461–468 had a higher phosphorus content than the control and similar results were also reported (K et al. 2002) on chickpea. Z-Y et al. (1997) observed that nodulation and plant growth were af- fected by the degree of mycorrhization, i.e. both were increased at higher levels. Interactions between the host and symbiont also varied with cultivars. K et al. (2002) concluded that mycorrhiza or Azoto- bacter or Rhizobium alone or their combinations could have an important effect on nodulation and nitrogen fixation in legumes. e principal effect of mycorrhiza on nodulation is P-mediated. e com- bined inoculations of symbionts showed significantly increased N-fixations growth and nutrient uptake in Leucaena leucocephala and Cajanus cajan (S et al. 1992). e mutual association accounted for better colo- nization and plant growth due to the interchange of carbon, phosphate and nitrogen between the host fungi and bacteria. According to B-E-D and M (1998), the dual inoculation of AM and Rhizobium had a synergistic effect on nodulation, plant growth, dry matter production and nitrogen fixation. Increased N-fixation due to increased mycorrhizal colonization in roots and nodulation may contribute to the growth and yield of plants (G, S 2002). J et al. (2001) reported that the growth of plants was enhanced by mycorrhizal infection by increasing nutrient uptake via an increase in the absorbing surface area of roots. Similar results were also obtained on wheat with plant growth promoting rhizobacteria ‘PGPR’ (K, Z 2007). D and T (1994) also observed similar results while using the soil-based AM inoculum. ey reported that the spore population was maximum in soil root based inoculum followed by AM spores inoculum. Re- cognition of the complexity of interactions among microbes in the rhizosphere has led to dual or co-inoculation of crops with both AM fungi and other rhizosphere microorganisms. By virtue of their interdependent and synergistic effects on P and N uptake, interactions between AM fungi and symbiotic N 2 -fixing bacteria are important when considering co-inoculations (G et al. 1985; R et al. 1986). Nodulation has been shown to be sometimes dependent on colonization by VAM fungi (A-A, B 1978). Similarly, inoculations with selected plant growth promoting rhizosphere bacteria have demonstrated synergistic benefits to the host when co-inoculated with VAM fungi. In many cases, colonization in the roots by AM fungi was increased by the other inoculants (A-A, B 1978; A et al. 1989). It has also been reported in this study that the ef- fect of AM fungi is increased when they were co- inoculated with other rhizosphere microflora like Rhizobium sp. and Trichoderma viride. CONCLUSION All double and triple inoculation (co-inoculation) treatments have a significant marked effect on the growth of A. catechu seedlings compared to the control sets in which a very small growth effect was observed. In double inoculation, the G. mosseae + Rhizobium sp. combination had a higher growth effect than the other treatments. In triple inocula- tion, G. mosseae + Rhizobium sp. + Trichoderma viride had a higher growth effect than the other treatments on A. catechu seedlings. is is due to the mutual positive action of Rhizobium sp. and AM fungi strains that helped in uptake of P and N from soil. On the other hand, Trichoderma viride being a biocontrol agent helped to control all pathogenic fungal attack in the rhizosphere of A. catechu. 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Relationships between fungal uptake of ammonium, fungal growth and nitrogen availability in ectomycorrhizal Pinus sylvestris seedlings. Mycorrhiza, 8: 215–223. ZHAO, YUHUA, TONGE M.M., WEIGUN L., 1997. Response of cowpea to mycorrhizal and rhizobial inoculation. Journal of Zhejiang Agricultural University, 23: 414–418. Received for publication December 24, 2008 Accepted after corrections April 28, 2009 Corresponding author: Dr. V P, M.Sc., Ph.D., Rain Forest Research Institute (ICFRE), Jorhat-785001, Assam, India tel.: + 943 557 0331, fax: + 376 239 5360, e-mail: bhardwajvpnpark@rediffmail.com Diverzita endomykorhizních hub a jejich synergistický efekt na růst Acacia catechu Willd. ABSTRAKT: Byla studována diverzita vesikulo-arbuskulárních mykorhizních (VAM) hub stromu Acacia catechu Willd. Spóry dominantních druhů VAM hub, bakterií Rhizobium sp. a houby Trichoderma viride byly izolovány z rizosféry stromu A. catechu a namnoženy v laboratoři. Hlavním cílem práce bylo studium vlivu vícenásobné ino- kulace VAM druhů Glomus mosseae a Glomus fasciculatum, směsi VAM druhů (Glomus spp. [kromě G. mosseae, G. fasciculatum ] s Acaulospora spp., Sclerocystis spp. a Gigaspora spp.), bakterií Rhizobium sp. a houby Trichoderma viride na růst semenáčků dřeviny A. catechu. Všechny očkované semenáčky vykázaly zlepšený růst proti kontrole, a to ve všech sledovaných parametrech (výška, čerstvá hmotnost a sušina kořenů a prýtů, počet spór VAM hub, procento mykorhizní kolonizace kořenů a počet hlízek bakterií Rhizobium). Příjem fosforu byl také proti kontrole vyšší. Výsledky prokázaly možnosti komerčního využití efektivních kmenů VAM hub společně s dalšími rizosferními mikroorganismy pro zajištění lepšího vzcházení a dalšího růstu semenáčků dřeviny A. catechu. Klíčová slova: Acacia catechu Willd.; vícenásobná inokulace; fosfor; Rhizobium sp.; Trichoderma viride; VAM diverzita . inoculation of a suitable mycorrhizal strain alone Diversity of endomycorrhizal fungi and their synergistic effect on the growth of Acacia catechu Willd. V. P 1 , A. A 2 1 Rain Forest. Re- cognition of the complexity of interactions among microbes in the rhizosphere has led to dual or co-inoculation of crops with both AM fungi and other rhizosphere microorganisms. By virtue of their. mind, the present study was under- taken to determine the effect of double and triple inoculation (co-inoculation) of AM fungi with other rhizospheric microflora on the growth performance of A. catechu. MATERIALS